ING: Introduction

Induction, Assay and Electrophoresis

Summary

This two-day experiment focuses on function of the lac operon. The consequences of the natural inducer, α-lactose, and the gratuitious chemical inducing agent, IPTG, on cell growth and expression of β-galactosidase will be studied. The effect of the antibiotic chloramphenicol will also be monitored during this experiment.

The experiment illustrates how to determine the specific growth rates of bacterial cultures. The fixed-time (single point) enzyme assay learned in the EZA experiment will be used to study the concepts of units of enzyme activity, volumetric activity, and specific activity. Linear and non-linear least squares fitting will be used to analyze the experimental results. Denaturing electrophoresis will also be used to study protein expression. Escherichia coli total cell lysates will be separated in an electrophoresis buffer containing sodium dodecyl sulfate and β mercaptoethanol. This method allows a substantial number of the proteins found in the bacterial cell to be separated. The molecular masses of abundant proteins can be determined from a standard curve prepared by comparison to the relative mobility of the molecular mass standards.

Background
For information on the lac operon:
Nelson & Cox. Lehninger Principles of Biochemistry, 4th Edition, pp. 1092-1101.

Introduction

Figure 1. The effect of an inducer on the synthesis of the proteins of the lac operon.

An operon is a coordinately regulated set of genetic information. The lac operon allows E. coli to rapidly begin using lactose present in the growth medium, as well as to rapidly stop transcription in the absence of lactose. FIGURE 1 contains a summary of the current understanding of the regulation of the lac operon, which represents one of the best-characterized examples of the genetic regulation of protein synthesis. One important early discovery was that a cytoplasmic protein called the lac repressor, LacI, inhibited expression of the lac operon in a medium lacking β substituted galactosides. In the absence of a suitable inducer, LacI binds to a nucleotide sequence on the DNA (called an "operator"), which is near to the nucleotide sequence where RNA-polymerase must bind to initiate transcription (called the "promoter"). Once bound to the DNA at the operator, LacI prevents transcription of the lac operon by inhibiting the binding of RNA-polymerase. As we will learn in the MBL experiments, the lac operon provides the basis for many of the commonly used bacterial expression vectors.

When lactose (D galactopyranose (β,1 → 4)-D-glucose) is present in the bacterial growth medium, it is metabolized to allolactose (D-galactopyranose (β,1 → 6)-D-glucose), the natural inducer. LacI binds the inducer, and causes a conformational that causes LacI to lose affinity for the operator. In the absence of the LacI-operator complex, rapid transcription of a polycistronic mRNA encoding the structural genes of the lac operon occurs. The mRNA is then translated into β-galactosidase, which cleaves lactose into D galactose and D glucose, galactose permease, which is involved in transporting lactose into the cell, and galactose acetylase, whose function is not known.

Figure 2. Isopropyl-1-thio-β-D-galactopyranoside (IPTG) is a commonly used gratuitous inducer for the lac operon.

One goal of this experiment will be to compare the cell growth and the induction of β-galactosidase activity in E. coli treated with the natural substrate, α-lactose, and a gratuitous inducer (i.e., one which cannot be metabolized). In this case, the gratuitous inducer will be isopropyl-1-thio-β-D-galactopyranoside (IPTG, FIGURE 2).

Denaturing Polyacrylamide Gel Electrophoresis

Summary
E. coli total cell lysates are separated in an electrophoresis buffer containing sodium dodecyl sulfate and β mercaptoethanol. This method allows a substantial number of the proteins found in the bacterial cell to be separated. The molecular masses of abundant proteins can be determined from a standard curve prepared by comparison to the relative mobility of the molecular mass standards.

Background

For information on denaturing gel electrophoresis:
Nelson & Cox. Lehninger Principles of Biochemistry
pp. 82-85—acid/base equilibria
pp. 92-96—electrophoresis
pp. 181-182—immunoblotting
pp. 296-298—DNA sequencing
p. 930-937—plasmid separation

Introduction

Gel electrophoresis is a powerful separation technique used for analyzing many biologically important molecules, including both proteins and nucleic acids. Since most biological polymers carry a net charge in solution, they will migrate in an electric field. The molecular migration is stabilized by polyacrylamide, agarose, or some other inert, solid support. Electrophoretic mobility can then be used to distinguish molecules that differ only slightly in net charge or shape, to detect amino acid residue changes in proteins, to determine protein molecular masses, or the size of polynucleotide fragments. This laboratory exercise focuses on protein elec¬trophoresis: nucleic acid electrophoresis will be discussed and applied in the molecular biology laboratory exer¬cise.

The function of sodium dodecyl sulfate and b-mercaptoethanol
An important application of protein electrophoresis requires the use of the denaturing de¬tergent sodium dodecyl sulfate (SDS) and a reducing agent. At neutral pH in 1% SDS and 0.1 M β mercaptoethanol, most protein molecules unfold, and inter- and intra-chain disulfide linkages are reduced. All higher order protein structure is therefore lost, and multisubunit proteins separate into their constituent subunits. In the random coil configuration, each protein chain is coated with a layer of SDS molecules. The negatively charged sulfate groups of the detergent, which usually far outnumber the charged groups belonging to the protein side chains, are exposed to the aqueous medium. Hence, the presence of SDS confers a large negative charge to the protein-detergent complex. Moreover, SDS is bound to most proteins in a nearly constant ratio (about l.4 g SDS per g protein), which provides a nearly constant charge to mass ratio. Consequently, proteins treated with SDS and β mercaptoethanol and then subjected to electrophoresis migrate toward the anode at a rate that is determined by the mass of the polypeptide, the porosity of the gel, and the electric field potential. In general, a small molecular mass polypeptide will have a faster rate of migration than a larger one. This observation forms the basis for using SDS polyacrylamide gels to characterize mixtures of proteins based on molecular mass.

Gels of a given porosity (achieved by ad¬justing the acrylamide and crosslinker concentrations) are calibrated with standard proteins of known molecular masses. The molecular mass of an unknown polypeptide can then be estimated from a standard curve for the given gel system.

Discontinuous gel electrophoresis
An important refinement of electrophoresis is the method of discontinuous ("disc") -gel elec¬trophoresis. A discontinuous gel system consists of two acrylamide gels; one gel is cast directly on top of the other. The lower gel (which is poured first and allowed to polymerize) is called the resolving gel. The resolving gel is so called because resolution takes place in this gel on the basis of size (due to sieving action of the polyacrylamide). The resolving gel contains an acrylamide concentration ranging from 5-30%, depending on the range of protein molecular masses to be separated. The higher the percentage of acrylamide used, the lower the molecular weight range that will be resolved. The upper gel, called a stacking gel, contains a much lower percentage of acrylamide than the resolving gel (usually about 2-3%), and offers little separation based on molecular mass. Instead, the stacking gel compresses the protein sample into a narrow band before it enters the resolving gel and thereby improves the resolution of the resolving gel. The stacking phenomenon is provided by differences in the pH and ionic strength of the buffers in the stacking gel, the resolving gel, and the buffer reservoir. Therefore, understanding the Henderson-Hasselbalch equation and the properties of buffers are essential to understand the functioning of discontinuous gel electrophoresis.

The stacking phenomenon
The protein bands are stacked as follows. At the pH of the stacking gel (6.8), the glycine amino group is predominantly in the protonated form, so that the amino acid is an electronically neutral zwitterion. In contrast, protein coated with SDS has a net negative charge. In this state, protein has an electrophoretic mobility between that of the glycine zwitterion (not migrating) and Cl- (the fast-moving counter-ion in the disc-gel buffer). The Cl- ion migrates rapidly primarily because it is small, and is followed by SDS-coated protein (having a large size, but also a large net charge), fol¬lowed by glycine (which has low net charge due to the pH equilibrium). As the Cl- ion moves ahead of the glycine, a drop in conductivity occurs at the inter¬face between the Cl- ions and the glycine ions. This decrease in conductivity gives rise to a voltage gradient that accelerates the slower moving protein and glycine. Ions ahead of the glycine-chloride interface experience are not exposed to a voltage gradient because the conductivity is not affected in this region; therefore, they are not accelerated. By continued acceleration into a zone of high conductivity, the protein band which sits between the chloride-glycine interface is stacked into a sharp band. The formation of a sharp band greatly improves the resolution that can be attained in the resolving gel. The larger the volume of the sample to be loaded, the more useful a stacking gel becomes.